Amino-acid based radicals are involved in a range of productive and destructive processes in living organisms. Functional, and typically highly controlled, radical chemistry occurs in enzymes that use amino acids as catalytically active redox cofactors. In contrast, oxidative stress conditions are well known to generate a range of uncontrolled, and for the organism, potentially harmful protein radical reactions. Despite the fact that the number of amino-acid radical enzymes and pathological conditions linked to dysfunctional amino-acid radical chemistry continuously increases, experimental characterization of parameters involved in protein radical reactions is still rudimentary. This situation reflects the simple fact that the characteristically reactive and thermodynamically hot radical state is highly challenging to study in the natural systems. The overall aim of the work described here is to use a library of well-structured model proteins specifically made to facilitate electrochemical, structural and spectroscopic studies of tyrosine and tryptophan radicals. During the proposed five-year funding period, we will structurally characterize the reduced and oxidized states of phenol-labeled proteins whose redox properties have already been determined by voltammetry techniques. Solution structures of reduced 2-, 3- and 4-mercaptophenol-13C will be obtained by NMR spectroscopy. The hydrogen-bonding interactions of the oxidized phenols in the 2- and 3-mercaptophenol-13C proteins will be determined by electron magnetic-resonance spectroscopy. The latter project involves cryo-trapping the phenol radicals using a specifically constructed UV/Vis absorption/photolytic spectrometer. The cryo-trapped radicals will be studied by CW and pulsed EPR, ENDOR and ESEEM spectroscopy at 9 and 94 GHz to determine the number and the strength of hydrogen bonds present between the protein matrix and the oxidized phenols. Once the structural studies have been completed, the redox properties of the mercaptophenol-13C proteins will be investigated by quantum chemical methods. Thus, experimental and calculated phenol potentials will be directly connected to protein structural motifs. This level of information is not available for any naturally occurring amino-acid radical. A similar set of voltammetry, structural and theoretical studies is proposed for a model protein enineered to contain a buried tyrosine-histidine hydrogen-bonded complex. Finally, the redox properties of tryptophan will be studied as a function of solvent exposure and electrostatic interactions. The short-term objective with the proposed experimental and theoretical studies is to correlate specific structural features with the reduction potentials and pKa values of tyrosine and tryptophan radicals in the model proteins. The long-term objective with the proposed work is to build a knowledge platform on which to quantitatively model the reduction potentials of functional and dysfunctional amino-acid radicals in natural systems. The studies described in this application have a clear potential to make a significant step towards realizing this long-term objective. PUBLIC HEALTH RELEVANCE: Protein radicals are involved in a range of both beneficial as well as harmful chemical reactions in living organisms. Little is know about these species since they are highly challenging to study experimentally in the natural systems. To increase our knowledge in this research area, we have developed a library of well- structured model proteins specifically made to study the chemical properties of protein radicals.